A) Microscopy—in Biological Research.
The development of fluorescent bio-molecular probes—especially fluorescent proteins enabling the observation of sub-cellular process and structure inside living cells, has come to pass as a renaissance in light microscopy. Now a variety of evolving bio-chemical techniques incorporate and/or are based upon combining fluorescent molecular probes and light microscopy. They provide both qualitative and quantitative visualization of specific molecular dynamics underlying live cellular activities. However, inasmuch as it is now clear that these biological processes depend upon spatio-temporal compartmentalization, the major focus for development in light microscopy, is to overcome certain systematic problems that obstruct our ability to render simple, yet quantitatively accurate three-dimensional mapping of fluorescent signals inside living cells (viz. 3-D microscopy).
Three Major Systematic Problems in 3-D Microscopy
The ability to render three-dimensional maps of fluorescent signals from micro objects visualized by light microscopy is limited mainly by three systematic artifacts.
a) Axial Aberration—the Elongation Effect
A central issue in light microscopy arises from limitations due to the spatial resolution of images. This is dependent upon the objective lens used and the geometry of the light focused by the lens. In general, microscope objectives with a high magnification and a high numerical aperture are used for achieving the best resolution (e.g. 63×; 100×/N.A. 1.4 oil immersion). However, there exist physical limitations for light collection through the glass lens of any objective. In particular, the xy resolution is always (at least two times) greater than in the z-axis (a.k.a “optical” axis). Specifically, x,y plane resolution is around 100-150 nm, whereas the resolution along the z-axis is much lower (around 300 to 500 nm), and this fact results in a major systematic artifact of light microscopy—i.e. “axial aberration”, whereby a spherical object at the focus of a microscope objective in fact appears to be elliptic in shape, with its largest extension along the z-axis, i.e. the light path. A schematic representation of this type of optical aberration (the “elongation” effect) is shown in FIG. 9. The axes of the microscope optics are x (9.1), y (9.2) and z (9.3). A feature 9.4 which is originally circular in reality appears to be elongated 9.5 due to optical aberration, and this problem is one of the major obstacles to overcome in 3 dimensional imaging microscopy.
b) Chromatic Aberration—Axial Misalignment of Multiple Wavelengths.
In addition to the problems of optical aberration and diminished axial resolution that reduce the ability to visualize in 3-D on a light microscope, another problem is referred to as “chromatic aberration” or “axial chromatic aberration”. Chromatic aberration occurs in applications where multi color images are acquired (Beyer). When performing z-scans at different wavelengths the light diffraction at the glass-to-medium-interface and within the whole microscope set-up depends upon the wavelength. As a consequence the focus for different wavelengths varies as a function of the z-axis displacement. Various types of correction are used to overcome chromatic aberration, for example, in confocal-microscopy (see below) a calibration of the “zero” position (unique to just one z-axis position) for all colors can be performed by an alignment of the light paths for the different wavelengths using multi colored (artificial) spatial-calibration samples (so-called “Focal Check Microparticles”). However, inasmuch as the calibration is unique to just one z-axis focal position, if a shift in focus occurs a systematic misalignment will again persist.
c) Out of Focus Light, and Diffraction Effects.
The third problem of three-dimensional fluorescence microscopy is that the micro-object itself comprises a complex three-dimensional form, and as such interferes with image visualization from the focal plane. This fact gives rise to a host of related problems for which there is no single general solution. Nonetheless, these problems are inextricably linked and must be considered critically in order to achieve true three-dimensional rendering. In general, these problems stem from light distortion caused by both the object itself, and the non-linear characteristics of light diffusion between different focal planes. These sorts of problem become critical in fluorescence microscopy where so-called “out-of-focus-light”, (light from parts of an object laying outside the focal plane) contribute to what is observed at the focal plane. This light compromises the crispness of the image, inasmuch as it introduces an out-of-focus “haze” into the image focal plane.
There are two ways of eliminating or reducing out-of-focus haze: i) confocal microscopy (see below), and ii) deconvolution. The latter is a calculation-intensive, algorithm-based mathematical method for sharpening images (from any source) that contain out-of-focus light. In general, the method requires that an axial stack of images be collected from the sample, at small (e.g. 50-500 nm) steps. The axial stack may then be processed using special algorithms that take into account a variety of optical parameters including the objective lens, and excitation/emission wavelengths. The corrected stack of images is then converted into a three-dimensional model of the object by either removing or reassigning the identified out-of-focus light (Egner, Markham). Using adequate graphics computing power, this three-dimensional model may then be rendered into animations allowing the object to be observed from any arbitrary viewing point.
Disadvantages of this approach are that it is calculation intensive (which costs time and processing power), and that it suffers inaccuracies and artifacts due to its extensive dependence upon calculation-based assumptions and/or corrections that must be applied at multiple stages during the processing procedure. It must also be noted that the best types of deconvolution algorithm rely upon prior measurement of a so-called PSF (Point-Spread-Function) specific to any given optical configuration (ie the microscope set-up). Put simply, the PSF is a measure of light diffusion from a sub-resolution point within a given focal plane, and can therefore be used to “re-map” out-of-focus light back into its appropriate 3-D voxel. However, a major problem with applied algorithms using PSF, is that it is extremely difficult to measure a “good” PSF. In particular, a major problem arises from the fact that the PSF in any given sample is itself altered as a function of the axial distance through the sample (Sedat). This fact results in any single PSF being representative only of a single focal plane, and therefore distorts reconstructions based upon z-axis stacks where clearly the z-axis is deflected in order to scan throughout the volume of a given sample. Finally, in addition to out-of-focus light, the sample (as stated above) alters the diffusion of light through its own volume by diffraction. This gives rise to a further group of problems, whereby light emission from the focal plane is deteriorated due to shading or diffraction do light by optically dense regions within the object itself (e.g. cell) that lay in between the microscope lens and the fluorescent features being imaged.
Advanced Techniques for Improved 3-D Fluorescence Microscopy
Three-dimensional imaging of micro objects requires that the above problems be addressed, and this may in part be achieved using numerous types of novel approach. Herein, a brief description summarises some of the advanced microscope techniques at the cutting edge of what is currently available. However, it should be noted that all will be improved substantially by the utility of the invention described herein. On this point, as for conventional fluorescence microscopy, all these techniques (without exception) achieve 3-D rendering by mechanically scanning the focus through multiple z-axis acquisitions at small (nm) intervals, collected from the sample volume. As such the micro object must, therefore, be immobilized by adherence to an optically transparent surface substrate (normally a 150 micron thick glass cover-slip). The resulting image “z-stack” must then be treated by calculation-intensive processing to yield 3-D rendering.
Single-photon excitation confocal fluorescence microscopy uses focused laser light for fluorescence excitation and, in general, a pinhole in the path of fluorescence emission, which allows in focus light derived from the x,y image plane to pass, but effectively rejects out-of-focus light. Fluorescence light measured using pinhole systems is detected using photo-multipliers and a scanning device. By way of an alternative, some commercial confocal systems use a so-called “Spinning-Disc” (Nipkow disk) system that achieves much the same result by rejecting out-of-focus light. However, the detection system differs inasmuch as it comprises a CCD camera, affording greater speed of acquisition. Either way, the advantage of confocal microscopy is that out-of-focus haze is greatly reduced, and by performing a z-scan, stacks of confocal images can be generated from a sample volume, in order to build a three-dimensional rendering of the imaged volume. Note that this approach still suffers from chromatic and axial aberration problems.
Multi-Photon Excitation Confocal Fluorescence Microscopy
A method for improving resolution in fluorescence microscopy is based upon the use of multi photon laser excitation. Fluorescence excitation of a fluorophore occurs at a certain wavelength λ nominally determined by its specific excitation absorption maxima. Efficient absorption of a single photon at this wavelength results in excitation and emission of fluorescent light (conventional fluorescence microscopy). However, excitation may also be achieved by simultaneous absorption of two photons of lower energy, displaying wavelengths approximately half the excitation maxima. This mode of so-called “multiphoton” excitation is considered to be “biphotonic or two-photon” induced fluorescence, and is made possible by grace of high energy pulsed lasers. In general this mode of excitation can be considered a means to excite fluorescence from, for example, a blue-green absorbing fluorophore using multi-photon excitation from a near-infra-red laser emitting sub-microsecond pulses of light. Inasmuch as the two photons of near-IR light are aligned and collide only at the focal plane of the optical set-up, the energy density of this multi photon excitation is concentrated solely at a single femtoliter volume within the microscope's focal plane. As such, multi-photon excitation is intrinsically confocal by nature. In effect this approach gives a pure, and efficient image free from “out of focus” fluorescence. The disadvantage of multi photon fluorescence microscopy is the requirement for high energy pulsed lasers to be attached to the microscope, resulting in high cost and large, difficult to manage equipment assemblage, maintenance and application.
4Pi Confocal (Theta) Microscopy, Standing-Wave Microscope (SWM), Incoherent Illumination Interference Image Interference Microscopy (I5M)
The generation of higher resolved three-dimensional images of cells can be improved by a combination of the techniques mentioned above and modifications of the optomechanic set-up. The use of two separate objective lenses for excitation and collection of fluorescence emission light leads to a smaller detection volume element and an equilateral resolution some 4 times higher than for conventional fluorescence microscopy (Egner). This technique is used in combination with multi photon fluorescence microscopy. In a 4Pi confocal fluorescence microscope two opposing microscope objective lenses are used to illuminate a fluorescent object from both sides and to collect the fluorescent emissions on both sides. Constructive interference of either the illumination wave fronts in the common focus or the detection wave fronts in the common detector pinhole results in an axial resolution approximately four times higher than in a confocal fluorescence microscope (Hell). The excitation/observation volume can be considerably decreased when the detection axis is rotated by an angle Theta (e.g. 90°) relative to the illumination axis as in Theta Microscopy (Lindek). Both methods bring along substantial limitations for the sample carrier and the microscope objective, which can be used. In addition there is a huge effort involved when aligning the two focal volumes of the objective lenses, which has to be done with sub-micrometer precision.
B) Micro Electrode/Fluidics Chamber(s) for Three Dimensional Manipulation of Micro Objects
Holding and lifting micro objects by negative dielectrophoresis in a well defined electric field minimum has been described since 1992 (Fuhr, G. et al. “Biochim. Biophys. Acta” 1108, 1992, 215-223). First, planar two-dimensional arrangements of micro electrodes have been used. They contained for example four electrodes with a tip-to-tip distance of 100 to 200 micrometers. Holding and lifting objects in these so-called “field traps” was only possible using alternating fields. Rotational fields had only limited trapping efficiency and were very sensitive to hydrodynamic streaming (Schnelle, Th. et al. “J. Electrostatics” 46, 1993, 13-28, Schnelle, Th. et al. “J. Electrostatics” 50, 2000, 17-29, Schnelle, Th. et al. “Appl. Phys. B” 70, 2000, 267-274, Reichle, Ch. et al. “Biochim. Biophys. Acta” 1459, 2000, 218-229). The development of so-called CellProcessors—three-dimensional electrode arrangements led to “field cages” consisting of eight electrodes and building up closed electric field cages (Schnelle, Th. et al., 1993, see above, Müller, Th. et al. “Biosensors & Bioelectronics” 14, 1999, 247-256, Reichle, Ch. et al. “Electrophoresis” 22/2, 2001, 272-282). “Cell Processors” containing dielectric field cages (DFCs) have been used in combination with a variety of high resolution optical techniques applied to micro objects, such as fluorescence correlation spectroscopy (FCS, Schnelle, Th. et al. “Electrophoresis” 21, 2000, 66-73), force measurements using laser tweezers (Fuhr et al. “Appl. Phys. A.” 67, 1998, 385-390), electro-rotation (Schnelle et al., see above), measurement of ligand-receptor binding forces (Reichle et al. 2001, see above) and confocal laser scanning microscopy (Müller, Th. et al. “European Biophysics Journal” 29/4-5, 2000, 12D-3 (Poster); Wissel, H. et al. “American Journal of Physiology Lung Cell Mol. Physiol.” 281, 2001, L345-L360).